PROJECT DESCRIPTION Salmonella enterica serovar Typhi (S. Typhi) and the related serovar S. Paratyphi cause typhoid fever in humans, a devastating disease that results in ~200,000 deaths every year. Although most of the cases occur in developing countries, outbreaks occasionally occur in the United States. Unlike other Salmonella enterica serovars, which can infect a variety of hosts and can cause limited gastroenteritis, S. Typhi is an exclusive human pathogen and causes systemic, often lethal, disease. Despite its Public Health importance, the mechanisms of pathogenesis of typhoidal Salmonellae remain poorly understood. Our laboratory has been exploring the unique aspects of S. Typhi pathogenesis and devoted a substantial amount of effort to the study of typhoid toxin, an A2B5 toxin that is highly conserved in typhoidal Salmonella serovars (i. e. S. Typhi and S. Paratyphi), but that it is largely absent from non-typhoidal Salmonellae. Typhoid toxin is an atypical AB toxin in that, unlike all known AB5 toxin family members, it has two enzymatically active subunits: an ADP ribosyl transferase (PltA) with an as of yet unidentified host target, and a deoxyribonuclease (CdtB), which inflicts DNA damage on intoxicated cells. These two subunits are covalently linked to one another and are associated to a homopentameric B subunit composed of PltB. Typhoid toxin is uniquely adapted to humans as it recognizes Neu5Ac-terminated sialoglycans on surface glycoproteins. Administration of typhoid toxin to experimental animals can reproduce many of the acute pathognomonic symptoms of typhoid fever, including stupor and lethargy, which most likely involve the central nervous system (CNS). Typhoid toxin exhibits a remarkable biology in that it is only produced by intracellular bacteria, and after its synthesis and assembly, it is released into the Salmonella-containing vacuole and subsequently transported to the extracellular space by specific vesicle transport carriers. During the past funding period we have unraveled many mechanistic aspects of the biology of typhoid toxin, including the description of its unique mechanism of intracellular expression, the characterization of all the steps of its remarkable transport pathways, the discovery of novel bacterial protein secretion mechanism, the description of its unique evolutionary history, and the discovery of an alternative form of typhoid toxin. Finally, these studies have led to the discovery of a novel cell-intrinsic pathogen restriction mechanism that prevents the replication of S. Typhi in mouse tissues and that it is antagonized by the mouse pathogen S. Typhimurium through the activity of specific type III protein secretion effectors absent from S. Typhi. These studies have raised very important questions related to pathogenesis of typhoid fever that we intend to pursue during the next funding period.